Article

Phenolic and Anthocyanin Compounds and

Antioxidant Activity of Tamarillo
(Solanum betaceum Cav.)
Tung Diep 1,2, Chris Pook 3 and Michelle Yoo 1,2*
1 School of Science, Faculty of Health and Environment Sciences, Auckland University of Technology,
Private Bag 92006, Auckland 1142, New Zealand; tung.diep@aut.ac.nz
2 Riddet Institute, Centre of Research Excellence, Private Bag 11 222, Palmerston North 4442, New Zealand

3 The Liggins Institute, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand;

chris.pook@auckland.ac.nz
* Correspondence: michelle.yoo@aut.ac.nz; Tel.: +64-9921-9999-6456

Received: 29 January 2020; Accepted: 15 February 2020; Published: 18 February 2020

Abstract: This study examined phenolics and anthocyanins present in Amber, Laird’s Large and
Mulligan cultivars of tamarillo that were cultivated in -Whangarei, Northland of New Zealand.
Samples were further separated by their tissue types, peel and pulp. Using LC-MS/MS, twelve
polyphenols were quantified and six (ellagic acid, rutin, catechin, epicatechin, kaempferol-3-
rutinoside and isorhamnetin-3-rutinoside) were detected for the first time in tamarillo. Mulligan
cultivar showed the highest amounts of phenolic and anthocyanin compounds and the highest
antioxidant activity. Phenolic compounds were mostly synthesized from shikimic acid route, and
chlorogenic acid dominated the profile regardless of cultivar and tissue types. Anthocyanin profile
was dominated by delphinidin-3-rutinoside in pulp. Higher amounts of anthocyanins were detected
in this study, which may be explained by favourable growth conditions (high light intensity and
low temperature) for anthocyanin biosynthesis in New Zealand. Higher antioxidant activity and
total phenolic content in peels than in pulps were found when assessed by Cupric Ion-Reducing
Antioxidant Capacity (CUPRAC), Ferric Reducing Ability of Plasma (FRAP) and Folin–Ciocalteu
assays, and a positive correlation (-r > 0.9, p ≤ 0.01) between the three assays was observed. Current
findings endorse that tamarillo has a great bioactive potential to be developed further as a functional
ingredient with considerable levels of antioxidant compounds and antioxidant activity.

Keywords: Tamarillo; phenolics; anthocyanins; antioxidant; chlorogenic acid; delphinidin-3-

rutinoside

1. Introduction
Tamarillo (Solanum betaceum Cav.) is a fruit species of family Solanaceae genus Solanum, which is
also known as tree tomato as its flesh closely resembles to that of the tomato [1]. As a subtropical
fruit, tamarillo is mainly grown in warmer and sheltered areas of the North Island in New Zealand
(Auckland and Hawkes Bay) [2]. New Zealand is one of the leading producers of tamarillo [3], with
the main export markets including America, Australia, Hong Kong, Singapore and Japan. The ripe
fruit turns to various colours (yellow, orange, red or purple) depending on the cultivars and exhibits
a slightly bitter, sour and astringent taste with a unique aroma [4]. In New Zealand, tamarillo are
available in yellow, red and purple-red cultivars, with the red being more popular and more common
than the others. Tamarillo is considered as underutilized fruit due to its texture, strong flavour and
some unidentified properties.
Phenolics (or polyphenols) and anthocyanins are secondary plant metabolites which are also
known as antioxidants. Health-promoting effects arising from prolonged high intake of polyphenols
Antioxidants 2020, 9, 169; doi:10.3390/antiox9020169 www.mdpi.com/journal/antioxidants
Antioxidants 2020, 9, 169 2 of 21

have been reported extensively in the literature, where reduction in risks of certain cancers,
cardiovascular diseases and diabetes and reduction in lipid oxidation and protective effects against
free radicals have been observed., Phenolics and anthocyanins have been shown to possess significant
antioxidant activity. For phenolic compounds, hydroxycinnamic acids (caffeic, ferulic, p-coumaric
and rosmarinic acids); hydroxybenzoic acids (gallic and vanillic acids); flavonol (kaempferol) and
flavanone (naringin) had been found in tamarillo from Ecuador, New Zealand [4] and Malaysia [5].
Anthocyanins are water-soluble pigments present in plants. They are commonly used as colourants
in the food industry. Antioxidant activity of anthocyanins is able to protect other food ingredients
against oxidation and help to maintain the nutritional value of foods. Presence of anthocyanins, such
as cyanidin, delphinidin and pelargonidin rutinosides in tamarillos from Brazil [6], Colombia [7],
Ecuador [4,8] and New Zealand [4], has been reported. The antioxidants are believed to partially
contribute to health-promoting effects of tamarillo, including antioxidation and antioxidative stress
[9], anti-obesity [10], anticancer [5,11] and anti-microbial activity [12], as well as protection against
lipid oxidation [13]. Variations arising from environmental factors (temperature, light intensity and
soil conditions) and post-harvest storage conditions contribute to variability in the presence of
antioxidants and their activities in tamarillos. Often, these are dependent on where they are
produced, even for the same cultivar. Scarce information on anthocyanin and phenolic compositions
of tamarillo, especially for those from New Zealand, are currently available, or data are several
decades old [1,14]. To our best knowledge, anthocyanin and phenolic profiles of tamarillo separated
by tissue types, into peel and pulp, have never been systematically investigated. The pulp of the
tamarillo is mostly consumed fresh, and the peel (skin) is discarded as waste.
In the present study, cultivars of Amber, Laird’s Large and Mulligan tamarillos grown in New
Zealand were explored for their phenolic and anthocyanin compositions, as well as antioxidant
activity, for the first time. Results from the current study will contribute to valorisation of tamarillo
as a good source of antioxidants and to propose potential benefits of the by-product (peels).

2. Materials and Methods

2.1. Materials
Three cultivars of ripe tamarillo fruit (21–24 weeks) were studied, including Amber (yellow),
Laird’s Large (red) and Mulligan (rich purple-red) from Northland, New Zealand. For each cultivar,
15 fruits were randomly selected to obtain representative samples, washed with running water and
then dried. Pulp and peel were manually separated, snap-frozen in liquid nitrogen, lyophilised and
ground to powder. Samples were packed and stored at −20 °C until analysis.

2.2. Chemicals, Reagents and Standards

All chemicals and reagents used were AnalaR grade or better. Acetone, acetonitrile, acetic acid,
methanol, isopropanol (IPA), formic acid, ethyl acetate, toluene and hydrochloric acid were obtained
from Thermo Fisher (Auckland, New Zealand). Folin-Ciocalteu reagent; neocuporine; copper (II)
chloride; 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) and Trolox (6-Hydroxy-2,5,7,8-
tetramethylchromane-2-carboxylic acid) were obtained from Sigma-Aldrich (Auckland, New
Zealand).
Analytical grade standards of polyphenols, including caffeic acid, (±) catechin, (−) epicatechin
and p-coumaric acid, were also purchased from Sigma-Aldrich (Auckland, New Zealand). Analytical
standards of other phenolics (gallic acid, ellagic acid, ferulic acid, chlorogenic acid, rutin, kaempferol,
kaempferol-3-rutinoside and isorhamnetin-3-rutinoside) and four anthocyanins (cyanidin-3-
glucoside, cyanidin-3-rutinoside, delphinidin-3-rutinoside and pelargonidin-3-rutinoside) were
obtained from Extrasynthese (Genay Cedex, France). Milli-Q water was produced by Purite Fusion
Milli-Q water purifying machine (Purite Limited, Thame, Oxon, UK).
Antioxidants 2020, 9, 169 3 of 21

2.3. Extraction and Quantification of Phenolics

A 50 ± 0.5 mg mass of dried, powdered sample was placed in an amber 1.8 mL glass vial, and a
1000 μL volume of 50% methanol was added. Samples were vortexed for 30 s and incubated in the
dark for 30 min at 4 °C with vortexing every 5 min. The samples were then centrifuged at 10,000 RCF
for 10 min at 4 °C to drive phase-transition. The supernatant was transferred to 1.5 mL polypropylene
Eppendorf tube and centrifuged again at 10,000 RCF for 10 min at 4 °C. The extract was separated
and transferred to a low-volume vial insert inside an amber 1.8 mL glass vial, then stored at –20 °C
until analysis.
The LC-MS/MS consisting of an Agilent 1260 Infinity Quaternary LC System (Santa Clara, CA,
USA) and an Agilent 6420 triple quadrupole mass spectrometer with multimode ionisation source
(model G1978B) was used to identify phenolic compounds in pulp and peel of tamarillo cultivars.
The phenolic compounds were separated along a Cortecs C18 column (2.1 × 100 mm, 2.7 μm; Waters,
-Tauton, Ireland). For mobile phase, 0.1% formic acid in Milli-Q for A and 0.1% acetic acid in
acetonitrile for B were used. LC gradient was set at: 0–12 min, 97% of A; 12–13.5 min, 75% of A; 13.5–
15.5 min, 10% of A and 15.5–23 min, 97% of A. Autosampler temperature was set at 4 °C. Injection
volume and flow rate were 3.0 μL and 0.25 mL/min, respectively. Mass spectrometer (MS) was run in
the negative mode with the total time of 23.0 min. The multimode (MMI) source operating at
electrospray ionization (ESI) parameters were gas temperature of 325 °C, gas flow of 6 L/min.
Capillary voltage and nebulizer were set to 2.0 kV and at 60 psi, respectively. - Vaporizer temperature
was set at 200 °C, and the column temperature was 25 °C.
Preliminary results showed that the retention characteristics of the peaks suggested the presence
of several phenolic compounds. Multiple reaction monitoring (MRM) was applied to detect and
quantify these analytes. Optimal MRM transitions of target compounds, including retention time,
precursor ion (m/z), product ion (m/z) and collision energy (V), are shown in Supplementary Table
S1. Chromatograms of standard injections are presented in Figure 1. Calibration curves of analyte
standards were constructed, and the linearity of these curves with at least six appropriate
concentrations was used for method validation. A summary of the standard curves for each
polyphenol with their associated regression equations, linear fit correlation coefficient (R2) and
calibration range are presented in Supplementary Table S1. The sensitivity of the method was
assessed by limit of detection (LOD) and limit of quantitation (LOQ). These two parameters were
calculated based on the standard deviation of the response and the slope from the standard curve
[15], and these are also presented in Supplementary Table S1.

2.4. Extraction and Quantification of Anthocyanins

Anthocyanins in the frozen, powdered tamarillo samples (50 ± 0.5 mg) were extracted with a
mixture of 50 μL of formic acid, 200 μL of isopropanol (IPA) and 400 μL of Milli-Q. The sample was
vortexed, followed by sonication in a water bath at 50 °C for 20 min. After addition of toluene (1.0
mL) into the solution to separate two liquid phases, the mixture was vortex-mixed again and
centrifuged at 10,000 RCF for 10 min. The lower, aqueous phase (60–80 μL) was placed in a clean
Eppendorf tube and centrifuged again at 10,000 RCF for 5 min. Then, 50 μL of the extract diluted with
50 μL of Milli-Q was placed into low-volume glass insert in a 1.8 mL brown glass autosampler vial.
The sample was kept at −20 °C until analysis.
LC-MS/MS coupled with fluorescence detection (FLD) and XSelect C18 column (2.1 × 100 mm,
3.5 µm; Waters, Tauton -, Ireland) were used to detect anthocyanin compounds. Flow rate, injection
volume and total run time were 0.4 mL/min, 1.0 μL and 9.0 min, respectively. The column
temperature was set at 25°C. The mobile phase A was 0.1% formic acid in acetonitrile and B was 0.6%
formic acid in Milli-Q. The elution gradient was set at 0–4 min, 18% of A; 4–9 min, 6% of A. Double
detection was implemented in a diode-array detector (DAD) at 520 nm as the preferred wavelength,
and the MS was run in the negative ion mode. The optimised MS program conditions operating at
ESI were gas temperature of 300 °C, gas flow of 6 L/min, vaporizer temperature of 300 °C, nebulizer
gas at 60 psi and capillary voltage of 2.2–2.5 kV.
Antioxidants 2020, 9, 169 4 of 21

Preliminary results showed that some of the peaks were coherent with cyanidin-3-glucoside,
cyanidin-3-rutinoside, delphinidin-3-rutinoside and pelargonidin-3-rutinoside based on their
retention characteristics by UV-spectrometry. Multiple reaction monitoring (MRM) transitions of four
analyte standards were optimised by Agilent MassHunter Optimizer software (Santa Clara, CA,
USA)), and data were collected by Agilent MassHunter Acquisition software. Chromatograms of
standard injections and optimal MRM parameters of analytes are shown in Figure 1 and
Supplementary Table S1, respectively. Quantification of anthocyanins in tamarillo extracts were
implemented using standard calibration curves fitted with at least six suitable concentrations
(Supplementary Figure S1). Method validation was carried out by assessing the linear fit correlation
coefficient (R2) of the linearity of standard curve. The LOD and LOQ were also calculated based on
the standard deviation of the response and the slope from the standard curve [15]. A summary of the
method validation for all of the identified anthocyanins is presented in Supplementary Table S1.

P
P3

P P

P2

P6
Ion counts

P9 A
A2

A1 A4
P1
P11
P8

P12

Time

Figure 1. Multiple reaction monitoring (MRM) chromatograms of 12 phenolics (P) and 4 anthocyanins
(A) standards. P1: gallic acid, P2: chlorogenic acid, P3: catechin, P4: caffeic acid, P5: epicatechin, P6: p-
coumaric acid, P7: ferulic acid, P8: ellagic acid, P9: rutin, P10: kaempferol-3-rutinoside, P11:
isorhamnetin-3-rutinoside, P12: kaempferol, A1: dephinidin-3-rutinoside, A2: cyanidin-3-glucoside, A3:
cyanidin-3-rutinoside and A4: pelargonidin-3-rutinoside.

2.5. Total Phenolic Content (TPC)

Total phenolic content (TPC) of tamarillo was identified by the Folin-Ciocalteu method reported
by Dorman, Koşar, Kahlos, Holm and Hiltunen [16], with some modifications. Firstly, 100 mg of
lyophilised sample was placed in a 15 mL centrifuge tube, and then 4 mL of 50% methanol was added.
The mixture was homogenised and left to rest for 60 min. The sample then was centrifuged at 1500
RCF for 15 min, and the extract from the top layer of the liquid was transferred into a 10 mL
volumetric flask. Then, the residue was extracted for a second time with 4 mL of 70% acetone, and
the steps described above, including homogenisation, resting and centrifugation, were followed. The
extract was transferred to volumetric flask containing the first extract, and then, Milli-Q was added
Antioxidants 2020, 9, 169 5 of 21

to the volumetric flask containing methanol and acetone extracts up to the 10 mL mark. From this, 1
mL from the volumetric flask was transferred to another volumetric flask and diluted to the 10 mL
mark using Milli-Q. This was used as the extracted sample solution.
The extracted sample solution (1 mL) was placed into a 10 mL glass vial, and then, 500 μL of
Folin-Ciocalteu reagent was added into the initial mixture and kept at room temperature for 5 min.
1.5 mL of 20% sodium carbonate (Na2CO3) solution was added to the vial, and the mixture was
incubated at room temperature for 120 min in the dark. The extracted sample solution was transferred
to a cuvette, and absorbance was measured at 765 nm against the blank using a UV-
spectrophotometer (Ultrospec 7000, Cambridge, England). The results were achieved by
interpolating the absorbances on a standard curve obtained with gallic acid (2.5–200 mg/L) under the
same conditions. Results were expressed as mg of gallic acid equivalent (GAE) per 100 gram of dry
weight (DW).

2.6. Antioxidant Activity by Ferric Reducing Ability of Plasma (FRAP) and Cupric ion-Reducing
Antioxidant Capacity (CUPRAC) Assays
FRAP assay was conducted according to Benzie and Strain [17], with modifications. Preparation
of the sample extract solution was the same as described in the TPC protocol. FRAP reagent (1:1:10,
v/v/v) was prepared by mixing FeCl3·6H2O (20 mM) with TPTZ (10 mM) in 40 mM HCl and acetate
buffer (300 mM, pH 3.6). The sample extract solution (100 μL) was mixed with Milli-Q (900 μL) and
the FRAP reagent (2 mL). Blank was prepared the same way, where the sample extract was replaced
with Milli-Q. Absorbance was measured at 593 nm on a UV-spectrophotometer (Ultrospec 7000) after
incubation at room temperature for 4 min. Quantification was carried out based on a Trolox standard
curve, generated from 5 to 160 mg/L. The antioxidant capacity of the extracts was expressed as μmol
Trolox equivalent antioxidant capacity per gram of dry weight (μmol TEAC/g DW).
The CUPRAC method published by Özyürek, Güçlü, Tütem, Başkan, Erçağ, Celik, Baki, Yıldız,
Karaman and Apak [18] was used, with some modifications. Similar sample preparation procedure
described in the TPC protocol was used. The sample extract solution (1 mL) was mixed with 1 mL of
CuCl2 (0.01M); 1 mL of ammonium acetate (1.0M, pH = 7.0); 1 mL of Neocuporine (0.0075M) in 96%
ethanol and 0.1 mL of Milli-Q to achieve the total solution volume of 4.1 mL. The reaction was left for
5 min, and the absorbance was then measured at 450 nm on a UV-spectrophotometer (Ultrospec 7000)
against MilliQ. A series of Trolox standard solutions in the range of 2.5–160 mg/L was used to produce
a standard curve, and the CUPRAC values were reported in μmol TEAC/g DW.

2.7. Statistical Analysis

Mean and standard deviation were calculated based on at least three independent measurements
(n ≥ 3) for each experiment. Two-way analysis of variance (ANOVA) and Fisher’s (LSD) multiple
comparison tests were applied to identify whether significant differences exist among different
cultivars (Amber, Laird’s Large and Mulligan) and tissues (peel and pulp) of tamarillo, together with
the interaction between these parameters. Pearson’s correlation coefficient was used to determine
correlation among total phenolic content and the two other antioxidant assays. Data analysis was
carried out using SPSS 25.0 (IBM Corp., Armonk, NY, USA), and the statistical significance level was
set at p < 0.05.

3. Results

3.1. Phenolic Compound Profiles

The LC-MS and the subsequent fragmentation of the predominant ion in MS-MS were used to
identify phenolic compounds from the aqueous methanol extracts of tamarillo. As shown in Figure
1, twelve mixed phenolic standards were successfully separated in the negative ion mode, and further
quantification of each identified polyphenol was carried out using a linear standard curve within a
serial concentration range. The first peak was not ideal, but it did not influence accuracy and precision
of the other compounds and the method. Good correlations of all the analysed phenolics were
Antioxidants 2020, 9, 169 6 of 21

achieved with R2 of the linearity > 0.99 (Supplementary Table S1). Additionally, the high sensitivity
of the chromatography system and the method was confirmed through very low LOD and LOQ,
which varied among different compounds. Rutin showed the lowest LOD and LOQ of 0.0092 and
0.0279 μg/L, respectively. Ellagic acid showed the highest LOD and LOQ of 1.7155 and 5.1984 μg/L,
respectively (Supplementary Table S1).
A total of 12 polyphenols were found and quantified in peel and pulp of three New Zealand
grown tamarillo cultivars. Among these phenolics, six compounds had been previously reported in
tamarillo [4,5,14]. Six other compounds were determined in tamarillo for the first time, and these
include ellagic acid, rutin, catechin, epicatechin, kaempferol-3-rutinoside and isorhamnetin-3-
rutinoside. Significantly (p < 0.05) different concentrations of phenolics were found between different
cultivars and tissues, as shown in Table 1. In the current study, chlorogenic acid (3-caffeoylquinic
acid) was the most abundant phenolic compound regardless of the cultivars and tissues. It ranged
from 54.67 to 278.03 mg/100 g DW, with higher amounts present in Mulligan and lower amounts in
Amber, as a general trend. Peels had more than three times of the chlorogenic acid concentration
compared to the pulps. The presence of chlorogenic acid in tamarillo has been reported by Wrolstad
and Heatherbell [14] and then later by Espin et al. [4] and Loizzo, Lucci, Núñez, Tundis, Balzano,
Frega, Conte, Moret, Filatova and Moyano [19]. Espin et al. [4] also reported chlorogenic acid as the
major phenolic compound in yellow and purple tamarillos from Ecuador and New Zealand, which
agrees with the findings of the current study for Amber and Mulligan. Previously reported
concentrations of chlorogenic acid in yellow and purple tamarillos from Ecuador was 25.04–42.73 and
50.33 mg/100 g DW, respectively, and in New Zealand purple cultivar, it was 163.62 mg/100 g DW
[4]. This phenolic compound was also dominant in tamarillo from Colombia, with 25.38 mg/100 g
DW in peel and 16.32 mg/100 g DW in pulp with seed [19]. These values were much lower than the
current findings from New Zealand tamarillo. Another study reported that, in Ecuadorian tamarillo,
the concentrations of caffeoylquinic acid and dicaffeoylquinic acid in the red type were 54.8 and 21.0
and, in yellow type, these were 32.8 and 17.1 mg chlorogenic acid equivalents per 100 g DW,
respectively [8].
Another three hydroxycinnamic acids, caffeic acid, ferulic acid and p-coumaric acid were found
in New Zealand tamarillos. Concentration of caffeic acid in peels was higher (approximately double)
than that in pulps for all of the three cultivars examined. By contrast, p-coumaric acid showed higher
concentration in pulps than in peels. The concentration of caffeic acid varied from 1.01 to 3.56 mg/100
DW, being the highest in Laird’s Large peel and the lowest in Amber pulp. In contrast, the Amber
pulp had the highest concentration of p-coumaric acid (0.12 mg/100 g DW), while the Laird’s Large
peel showed the lowest content of p-coumaric acid (0.02 mg/100 g DW). Caffeic and p-coumaric acids
were quantified in pulp of Malaysian tamarillo, with concentration of 0.165 and 0.041 mg/100 g DW,
respectively [5]. Loizzo et al. [19] quantified the concentration of p-coumaric acid in both peel and
pulp of Colombian tamarillo at 0.02 mg/100 g DW. These values from both studies were lower than
most of the current findings. Both Amber pulp and Laird’s Large pulp contained ferulic acid in
extremely low concentrations (<0.005 mg/100 g DW), whereas all other samples had trace amounts
ranging from 0.01 to 0.04 mg/100 g DW. Low concentrations (0.005 mg/100 g DW) of ferulic acid were
also observed by Mutalib et al. [5]. A higher concentration of this acid has been observed in peel and
pulp of tamarillo from Colombia with 0.87 and 0.76 mg/100 g DW, respectively [19]. Espin et al. [4]
reported the presence of ferulic acid dehydrodimers in two yellow cultivars (0.12 and 0.06 mg/100 g
DW) and the purple type tamarillo (3.27 mg/100 g DW) from Ecuador, as well as the purple-type
tamarillo from New Zealand (21.17 mg/100 g DW), in their study. The higher contents of
hydroxycinnamic acids in purple and red New Zealand tamarillos compared with the yellow one
were in agreement with the finding observed for red and yellow tamarillos from Ecuador [8]. It was
evident from our findings that hydroxycinnamic acids were one of the major polyphenolic groups in
tamarillo and accounted for more than 85% and 55% of the polyphenols in peel and pulp of the three
tamarillo cultivars, respectively (Figure 2).
Two hydroxybenzoic acids, gallic acid and ellagic acid were found in New Zealand-grown
tamarillo, which ranged from 0.9 to 1.09 mg/100 g DW (0.3–0.9%) (Table 1 and Figure 2). Both
Antioxidants 2020, 9, 169 7 of 21

hydroxybenzoic acids showed low concentrations in all analysed samples, though significant
differences between cultivars were observed (Table 1). The content of gallic acid was similar in both
peel and pulp of Amber cultivar (approximately 0.8 mg/100 g DW), while the pulp of Laird’s Large
and Mulligan had slightly higher gallic acid content than their peels (0.93 and 1.0 mg/100 g DW
compared with 0.79 and 0.8 mg/100 g DW, respectively) (Table 1). Gallic acid was firstly quantified
(0.302 mg/100 g DW) in Malaysian tamarillo by Mutalib et al. [5]. Despite being present in trace
amounts (about 0.1 mg/100 g DW), the presence of ellagic acid was detected in tamarillo for the first
time by the current study. All three cultivars in both peel and pulp showed relatively similar
concentrations of ellagic acid, 0.09 to 0.12 mg/100 g DW (Table 1). There was no significant difference
(p > 0.05) in ellagic acid content among three cultivars, and no significant interaction between
cultivars and tissues was found.
Apart from the two phenolic acid groups, one flavonol (kaempferol) was found in New Zealand-
grown tamarillo at a very low concentration, from 0.43 to 0.5 mg/100 g DW. All the peels had similar
concentrations of kaempferol, whereas the Amber pulp showed a slightly higher amount of this
phenolic than the Laird’s Large and Mulligan pulps, with significant differences observed (Table 1).
The percentage of this flavonol in the total phenolic concentration was 0.1–0.2% in peel and 0.4%–
0.5% in pulp (Figure 2). The concentration of this flavonol in Malaysian tamarillo has been reported
as 0.05 mg/100 g DW [5], which was lower than the value obtained in the current study.
Two flavanols, catechin and epicatechin, were reported for the first time in the current study.
These accounted for 0.7–1.3% and 1.7–5.4% in peels and pulps of all phenolics, respectively (Figure
2). Amber showed significantly higher concentration of catechin in both peel (2.13 mg/100 g DW) and
pulp (3.91 mg/100 g DW) than the two other cultivars. A low amount of catechin was detected in
Laird’s Large and Mulligan cultivars (0.28–0.33 mg/100 g DW), as well. Significant differences (p <
0.05) were observed in catechin content among cultivars and tissues, with significant interactions
observed between cultivars and tissues (Table 1). By contrast, the Mulligan had the highest content
of epicatechin in both peel (2.6 mg/100 g DW) and pulp (2.31 mg/100 g DW) (Table 1). Additionally,
peels and pulps of Amber and Mulligan cultivars showed relatively similar concentration of this
flavanol, whereas the Laird’s Large peel had slightly lower amounts of epicatechin than the pulp
(Table 1). There were no significant differences (p > 0.05) in epicatechin content between pulps and
peels, and no significant interaction between cultivars and tissues was found.
Presence of flavonol glycosides has never been reported in tamarillo before. Flavonol glycosides
comprised 8.3–12.5% of all phenolics in peels and 35.3–41.8% of all phenolics in pulps (Figure 2).
Rutin, known as quercetin-3-rutinoside, dominated the flavonol glycoside profile for peels of Amber
and Mulligan cultivars by being the second-most abundant polyphenol (Table 1). The concentrations
of rutin were significantly different among all tamarillo cultivars and tissues (p < 0.05), with higher
concentrations observed in peels than in pulps (approximately 9.6, 7.5 and 3.8 times more for
Mulligan, Amber and Laird’s Large cultivars, respectively). Among different tissues, Amber showed
the highest content of rutin, with 24.33 and 3.23 mg/100 g DW in peel and pulp, respectively (Table
1). The current study reports kaempferol-3-rutinoside as the second-most abundant phenolic in pulps
of all cultivars, as well as in Laird’s Large peel. Significant differences in the content of kaempferol-
3-rutinoside were observed among six samples (p < 0.05) (Table 1). Laird’s Large pulp and Amber
peel showed the highest and the lowest concentrations of kaempferol-3-rutinoside, with 50.04 and
8.32 mg/100 g DW, respectively. For pulp, it was approximately 2.6–3.6 times higher than that in the
peel across all the cultivars (Table 1). Trace amount of isorhamnetin-3-rutinoside was found in pulp
of all cultivars (≤0.16 mg/100 g DW). Among all peel samples, Laird’s Large had the highest
concentration (0.96 mg/100 g DW), followed by Mulligan (0.9 mg/100 g DW) and then Amber (0.59
mg/100 g DW) (Table 1).
The total concentration of phenolic compounds detected in the peels was slightly more than a
double of the pulps for all cultivars, as shown in Table 1. For both peel and pulp, Mulligan showed
the highest total phenolics (TPs). The TPs in Laird’s Large pulp were slightly higher than the Amber
pulp, whereas the Amber peel and Laird’s Large peel had relatively similar TPs (Table 1). Previous
Antioxidants 2020, 9, 169 8 of 21

findings in Colombian tamarillo showed that TPs in peel and pulp were 28.41 and 20.72 mg/100 g
DW [19], which were lower than our findings.
Antioxidants 2020, 9, 169 9 of 21

Table 1. Profiles of phenolics and anthocyanins (mg/100 g DW) in three tamarillo cultivars, separated by cultivars and tissues. Values are expressed as Mean ± SD (n = 4).

Peel Pulp -p-value*

Bioactive Compounds
Amber Laird’s Large Mulligan Amber Laird’s Large Mulligan C T C×T
Chlorogenic acid 225.85 ± 12.43ax 231.18 ± 9.76bx 278.03 ± 11.89cx 54.67 ± 3.81ay 66.35 ± 1.1by 73.95 ± 1.98cy < 0.05 < 0.05 < 0.05
Caffeic acid 2.61 ± 0.18ax 3.56 ± 0.52bx 3.52 ± 0.85bx 1.01 ± 0.03ay 1.2 ± 0.06by 1.32 ± 0.04by < 0.05 < 0.05 0.0328
p-coumaric acid 0.05 ± 0.01ax 0.02 ± 0.01bx 0.03 ± 0.01cx 0.12 ± 0.01ay 0.07 ± 0.01by 0.08 ± 0.01cy < 0.05 < 0.05 < 0.05
Ferulic acid 0.03 ± 0.03ax 0.01 ± 0.01bx 0.04 ± 0.03cx < 0.005ay < 0.005by 0.02 ± 0.02cy < 0.05 < 0.05 0.1158
Total hydroxycinnamic acids 228.53 ± 12.65ax 234.77 ± 10.3bx 281.63 ± 12.78cx 55.80 ± 3.86ay 67.62 ± 1.18by 75.37 ± 2.04cy < 0.05 < 0.05 < 0.05
Gallic acid 0.8 ± 0ax 0.79 ± 0bx 0.8 ± 0.01bx 0.79 ± 0ay 0.93 ± 0.06by 1 ± 0.2by < 0.05 < 0.05 < 0.05
Ellagic acid 0.12 ± 0.04x 0.11 ± 0.03x 0.1 ± 0.04x 0.11 ± 0.02y 0.09 ± 0.01y 0.09 ± 0.02y 0.2304 0.0483 0.6023
Total hydroxybenzoic acids 0.91 ± 0.05ax 0.9 ± 0.03abx 0.91 ± 0.05bx 0.90 ± 0.03ay 1.01 ± 0.07aby 1.09 ± 0.22by 0.0227 < 0.05 0.0147
Kaempferol 0.43 ± 0.01ax 0.43 ± 0.01bx 0.43 ± 0.01bx 0.5 ± 0.04ay 0.45 ± 0.01by 0.45 ± 0.01by < 0.05 < 0.05 < 0.05
Total flavonols 0.43 ± 0.01ax 0.43 ± 0.01bx 0.43 ± 0.01bx 0.5 ± 0.04ay 0.45 ± 0.01by 0.45 ± 0.01by < 0.05 < 0.05 < 0.05
Catechin 2.13 ± 0.83ax 0.28 ± 0bx 0.33 ± 0.02bx 3.91 ± 0.68ay 0.3 ± 0by 0.32 ± 0by < 0.05 < 0.05 < 0.05
Epicatechin 1.36 ± 0.03a 1.49 ± 0.12b 2.6 ± 0.72c 1.34 ± 0a 1.73 ± 0.01b 2.31 ± 0.02c < 0.05 0.763 0.0545
Total flavanols 3.49 ± 0.86ax 1.78 ± 0.13bx 2.94 ± 0.74cx 5.25 ± 0.68ay 2.03 ± 0.01by 2.63 ± 0.02cy < 0.05 < 0.05 < 0.05
Rutin 24.33 ± 1.85ax 3.71 ± 0.45bx 12.68 ± 1.1cx 3.23 ± 0.08ay 0.97 ± 0.03by 1.32 ± 0.03cy < 0.05 < 0.05 < 0.05
Kaempferol-3-rutinoside 8.32 ± 0.57ax 19.22 ± 1.2bx 12.45 ± 1.09cx 30.72 ± 0.97ay 50.04 ± 1.12by 45.6 ± 1.41cy < 0.05 < 0.05 < 0.05
Isorhamnetin-3-rutinoside 0.59 ± 0.05ax 0.96 ± 0.05bx 0.9 ± 0.02cx 0.16 ± 0.01ay 0.13 ± 0by 0.14 ± 0cy < 0.05 < 0.05 < 0.05
Total flavonol glycosides 33.25 ± 2.47ax 23.89 ± 1.7bx 26.03 ± 2.21bx 34.11 ± 1.06ay 51.14 ± 1.14by 47.06 ± 1.44by < 0.05 < 0.05 < 0.05
Total phenolics 266.62 ± 16.04ax 261.77 ± 12.16bx 311.93 ± 15.80cx 96.56 ± 5.67ay 122.26 ± 2.42by 126.60 ± 3.74cy < 0.05 < 0.05 < 0.05
Delphinidin-3-rutinoside 0.43 ± 0.02ax 32.41 ± 2.98bx 49.11 ± 2.23cx 29.17 ± 2.47ay 254.76 ± 6.33by 273.36 ± 12.7cy < 0.05 < 0.05 < 0.05
Cyanidin-3-glucoside n.d 0.33 ± 0.04a 1.97 ± 0.14b n.d n.d n.d < 0.05 – –
Cyanidin-3-rutinoside 0.29 ± 0ax 68.72 ± 4.33bx 114.47 ± 5.97cx 0.18 ± 0.02ay 25.94 ± 1.99by 30.67 ± 2.76cy < 0.05 < 0.05 < 0.05
Pelargonidin-3-rutinoside 0.52 ± 0.06ax 54.36 ± 3.24bx 93.63 ± 2.48cx 0.35 ± 0.03ay 200.66 ± 8.51by 182.81 ± 11.17cy < 0.05 < 0.05 < 0.05
Total anthocyanins 1.24 ± 0.08ax 155.82 ± 10.58bx 259.18 ± 10.81cx 29.70 ± 2.52ay 481.37 ± 16.83by 486.84 ± 26.63cy < 0.05 < 0.05 < 0.05
n.d: not detected. * Statistical significance for cultivar (C), tissue (T) and the interaction of both types (C × T). Means shown in a, b, c are significantly different at p < 0.05
between cultivars. Means shown in x, y are significantly different at p < 0.05 between tissues. SD values of less than 0.004 are presented as 0.
Antioxidants 2020, 9, 169 10 of 21

Hydroxycinnamic acids Flavonol glycoside Flavanols Hydroxybenzoic acids Flavonols

2.1 0.9
0.4
Mulligan pulp 59.5 37.2

1.7 0.8 0.4

Laird's Large pulp 55.3 41.8

5.4 0.9
0.5
Amber pulp 57.8 35.3
0.3
0.9 0.1
Mulligan peel 90.3 8.3
0.3
0.7 0.2
Laird's Large peel 89.7 9.1
0.3
1.3 0.2
Amber peel 85.7 12.5

0% 20% 40% 60% 80% 100%

Figure 2. Proportion (%) of five dominating phenolic classes separated by tissue and cultivar types of
tamarillo.

3.2. Anthocyanin Compound Profiles

Four anthocyanins were captured and quantified in peels and pulps of New Zealand-grown
tamarillos using LC-MS/MS. All data acquired for method validation are shown in Supplementary
Table S1 and Figure S1 with R2 > 0.999, as well as low LOD and LQD values. A representative MRM
transitions chromatogram of the determined anthocyanins in tamarillo extracts and their anthocyanin
profiles are illustrated in Figure 1 and Table 1, respectively. To our knowledge, this was the first
attempt to quantify anthocyanins in two different tissues of tamarillo. Cyanidin-3-glucoside was
quantified for the first time in Laird’s Large and Mulligan cultivars, although these were only
detected in the peels, at 0.33 and 1.97 mg/100 g DW, respectively. Pulps showed higher total
anthocyanin concentration than the peels in all cultivars. Additionally, Laird’s Large and Mulligan
cultivars had higher concentrations of each anthocyanin than Amber for both peel and pulp tissues
(Table 1). The proportion of different anthocyanins was relatively similar in Laird’s Large pulp and
Mulligan pulp.
There was a huge variation in the total anthocyanin content of peels. For Mulligan, Laird’s Large
and Amber, 259.18, 155.82 and 1.24 mg/100 g DW were found, respectively. Compared to the peels
of other fruits with similar colour (red), the concentration of total anthocyanins was evidently higher
in tamarillo. For example, the peels of red grape, red plum and red apple had the total anthocyanin
concentrations of 27, 20 and 12 mg/100 g FW, respectively (approximately equal to 138.75, 156.62 and
83.1 mg/100 g DW, respectively) [20]. Total anthocyanin content in pulp of Mulligan, Laird’s Large
and Amber was much higher than its counterpart (peel), with 486.84, 481.37, and 29.70 mg/100 g DW,
respectively. The total anthocyanins in the pulp of tamarillo were higher compared to the pulp of
strawberry, grape and cranberry, with 41.09, 28.09 and 19.30 mg/100 g FW, respectively
(approximately equal to 454.03, 144.35 and 152.21 mg/100 g DW, respectively) [21].
Significant differences in individual and total anthocyanin concentrations were found among
different cultivars and tissues. The interaction between cultivars and tissues were significant (p < 0.05)
for anthocyanins (Table 1). Presence of cyanidin 3-rutinoside, delphinidin-3-rutinoside and
pelargonidin 3-rutinoside have been previously reported in tamarillo [4]. Delphinidin-3-rutinoside
was the most dominant anthocyanin in Laird’s Large and Mulligan pulps (254.76 and 273.36 mg/100
g DW, respectively), followed by pelargonidin 3-rutinoside and cyanidin 3-rutinoside. For peels of
Antioxidants 2020, 9, 169 11 of 21

Laird’s Large and Mulligan, cyanidin-3-rutinoside was the most abundant anthocyanin; 68.72 and
114.47 mg/100 g DW, respectively (Table 1). Previously, cyanidin 3-rutinoside has been reported as
the main pigment in peel of New Zealand purple variety [14], and delphinidin-3-rutinoside being the
major anthocyanin in the pulp of the purple variety [4,14]. For Laird’s Large and Mulligan,
delphinidin-3-rutinoside and pelargonidin-3-rutinoside showed significantly higher concentrations
in pulps than in peels by 5.6–7.8 and 2.6–3.7 times more, respectively. By contrast, peels had higher
concentrations of cyanidin-3-rutinoside than pulps by 2.0 and 3.7 times for Laird’s Large and
Mulligan, respectively (Table 1).
Compared to Mulligan and Laird’s Large, which carry purple and red colours, Amber cultivar,
carrying yellow colour, possessed the least amounts of anthocyanins. To date, the presence of
anthocyanins has never been reported in New Zealand Amber cultivar or yellow variety from other
sources. The major anthocyanin in Amber peel and pulp was pelargonidin-3-rutinoside (0.52 mg/100
g DW) and delphinidin-3-rutinoside (29.17 mg/100 g DW), respectively (Table 1). Concentrations of
cyanidin-3-rutinoside, delphinidin-3-rutinoside and pelargonidin-3-rutinoside in Laird’s Large and
Mulligan tamarillo in this study were significantly higher than that reported in the red variety from
Ecuador [8] and in the purple cultivars from Ecuador and New Zealand [4]. Additionally, the New
Zealand Laird’s Large and Mulligan tamarillos had greater total anthocyanin content (481.37 and
486.84 mg/100 g DW, respectively) than Brazilian tamarillo, with 8.5 mg/100 g FW (approximately
equal to 70.83 mg/100 g DW, with estimated moisture of 88%) [6]; Ecuadorian tree tomato, with 165.1
mg/100 g DW [8] or 102.35 mg/100 g DW [4] and New Zealand purple cultivar from the previous
study (168.88 mg/100 g DW) [4].

3.3. Total Phenolic Content (TPC)

As shown on Table 2, peels had significantly higher (p < 0.05) TPC and antioxidant capacity than
pulps. From the three cultivars studied, Mulligan was the most bioactive (p < 0.05), with relatively
high TPC (2225.06 and 874.09 mg GAE/100 g DW), as well as strong antioxidant activities, as
determined by CUPRAC assay (265.29 and 71.57 μmol TEAC/g DW) and FRAP assay (161.74 and
72.14 μmol TEAC/g DW) for peel and pulp, respectively (Table 2).
The TPC of peels and pulps varied among tamarillo cultivars, where the values were almost a
double in peels compared to that of the pulps. The phenolic content was the highest in Mulligan peel
(2225.06 mg GAE/100 g DW) and the lowest in Amber pulp (678.98 mg GAE/100 g DW). The purple
and red cultivars have been reported to have greater values of phenolic contents than the yellow ones
[1,8,22]. The TPC of red and gold tamarillos from New Zealand were 190.8 and 116.6 mg GAE/100 g
FW (1564 and 1060 mg/100 g DW), respectively [1]. These were higher than the values in the current
study. Vasco et al. [22] reported that the TPC of golden-yellow and purple-red tamaillo cultivars from
Ecuador were 78 and 113 mg GAE/100 g FW (557 and 1413 mg/100 g DW), respectively.

3.4. Antioxidant Activity

There are several assays to assess antioxidant activity in vitro, where CUPRAC and FRAP
methods are the most widely used. Significant differences (p < 0.05) in the antioxidant activity were
found among different cultivars and tissues, individually. A significant interaction between the
cultivars and tissues was also observed. Antioxidant capacity, presented by the CUPRAC value, was
significantly higher in Mulligan than in Laird’s Large and Amber cultivars for peels (265.29 compared
with 136.68 and 117.59 μmol TEAC/g DW, respectively) and pulp (71.57 compared to 52.42 and 42.92
μmol TEAC/g DW, respectively) (Table 2). The CUPRAC values for peels were approximately 3.7, 2.8
and 2.6 times higher than pulps for Mulligan, Amber and Laird’s Large cultivars, respectively.
Similar phenomena were observed when assessed with the FRAP assay. Mulligan showed the
strongest anti-radical efficacy in both tissues, and peel owned higher antioxidant ability than pulp
for all of the cultivars. The FRAP values varied from 52.23 to 161.74 μmol TEAC/g DW, with the
highest value found in Mulligan peels and the lowest in Amber pulp. The FRAP values for pulp were
1.6–2.2 times less than that for the peels. Espin et al. [4] have reported the FRAP value for pulp of
purple tamarillo from New Zealand as 50 μmol TEAC/g DW [4], which was lower than the current
Antioxidants 2020, 9, 169 12 of 21

study. They have also reported the FRAP values for the pulp of yellow and purple tamarillos from
Ecuador as 10–17 and 15 μmol TEAC /g DW, respectively [4].

Table 2. Total phenolic content and antioxidant activity of tamarillo separated by cultivars and
tissues. Values are expressed as Mean ± SD (n = 4).

Total Phenolic Content CUPRAC Value FRAP Value

Tissues Cultivars
(mg GAE/100 g DW) (μmol TEAC/g DW) (μmol TEAC/g DW)
Amber 1583.8 ± 40.09ax 117.59 ± 9.35ax 84.7 ± 11.13ax
Peel Laird’s Large 1673.28 ± 63.97bx 136.68 ± 6.72bx 102.55 ± 12.19bx
Mulligan 2225.06 ± 50.87cx 265.29 ± 18.35cx 161.74 ± 14.53cx
Amber 678.98± 19.09ay 42.92 ± 8.73ay 52.23 ± 6.7ay
Pulp Laird’s Large 707.04 ± 30.65by 52.42 ± 8.39by 60.19 ± 5.21by
Mulligan 874.9 ± 30.48cy 71.57 ± 7.81cy 72.14 ± 9.41cy
C < 0.05 < 0.05 < 0.05
p-value* T < 0.05 < 0.05 < 0.05
C×T < 0.05 < 0.05 < 0.05
* Statistical significance for cultivar (C), tissue (T) and the interaction of both types (C × T). Means
shown in a,b,c are significantly different at p < 0.05 between cultivars. Means shown in x,y are
significantly different at p < 0.05 between tissues. FRAP: Ferric Reducing Ability of Plasma and
CUPRAC: Cupric Ion-Reducing Antioxidant Capacity.

3.5. Correlation between TPC, CUPRAC and FRAP

Results from Pearson’s correlation coefficient revealed a strong correlation between TPC and
antioxidant activity. The correlation between TPC–CUPRAC and TPC–FRAP were 0.941 (p < 0.01)
and 0.906 (p < 0.01), respectively (-Table 3). The high correlation between TPC and antioxidant activity
(determined by DPPH method) of tamarillo had been previously reported by Acosta-Quezada,
Raigon, Riofrio-Cuenca, Garcia-Martinez, Plazas, Burneo, Figueroa, Vilanova and Prohens [23] for r
= 0.8607 and by Mutalib et al. [11] for r = 0.998. Vasco, Ruales and Kamal-Eldin [24] reported the
correlation between the TPC and FRAP value of 17 Ecuadorian fruits, including purple-red and
golden-yellow tamarillos, as 0.62. Similarly, the results of the two antioxidant assays (CUPRAC and
FRAP) were highly correlated (r = 0.959, p < 0.01) (Table 3). For 17 Ecuadorian fruits, including
tamarillo, a correlation of between DPPH and FRAP assays was 0.908 [24].

Table 3. Correlation between total phenolic content (TPC) and antioxidant activity (measured by
CUPRAC and FRAP assays) of three tamarillo cultivars.

Content TPC CUPRAC FRAP

Pearson’s Correlation 1 0.941** 0.906**
TPC Sig. (2-tailed) 0.000 0.000
Number 24 24 24
Pearson’s Correlation 0.941** 1 0.959**
CUPRAC Sig. (2-tailed) 0.000 .000
Number 24 24 24
Pearson’s Correlation 0.906** 0.959** 1
FRAP Sig. (2-tailed) 0.000 0.000
Number 24 24 24
** Correlation is significant at the 0.01 level (2-tailed).

4. Discussion
Three tamarillo cultivars from New Zealand were analysed for phenolic and anthocyanin
components and assessed for antioxidant capacity. In the literature, the characteristics of Amber and
Mulligan cultivars, which are also known as yellow and purple varieties, respectively, are rarely
found. Most of the reported studies in tamarillo have focused on Laird’s Large cultivar, which is also
known as red variety, as it is the most commonly grown and consumed tamarillo cultivar.
Antioxidants 2020, 9, 169 13 of 21

Variations in the profiles of phenolic and anthocyanin compounds in tamarillo between the
current study and the previously reported values in the literature may be partly explained by climate
and environmental factors, including stronger UV light, dry soils and lack of rain in Northland of
New Zealand where the tamarillos for the current study were sourced from. Clean and green
environment of New Zealand and their breeding and pest management techniques may have
provided ideal conditions for tamarillo cultivation, compared to other countries. Variations may also
have risen from the cultivars of fruit, post-harvest handling, storage period, ripeness of the fruit and
extraction and analytical methods for quantification. For example, Mertz et al. [8] used aqueous
acetone containing 2% formic acid as extraction solvent for anthocyanin quantification, and Espin et
al. [4] used a combination of methanol and 0.1% formic acid for extraction with 0.1% trifluoracetic
acid and acetonitrile as mobile phases. In this study, a mixture of formic acid, Milli-Q, IPA and
toluene was used as extraction solvent, with a mobile phase of 0.1% formic acid in acetonitrile and
0.6% formic acid in Milli-Q. A toxicity problem can be caused by using organic solvents (methanol or
ethanol) for anthocyanin extraction [25]; therefore, water-based extracts was used. Additionally, we
used pure formic acid to lower the pH of the solution at which these anthocyanins are more stable.
The formation of flavylium cation at acidic condition is known to significantly enhance the solubility
of anthocyanins in water [25]. IPA is a good nontoxic alternative to preserve biological specimens; in
this case, anthocyanins. Toluene was used to separate different layers to enhance the purity of the
extracts. The use of LC-MS/MS for quantification of these compounds may have helped in identifying
those at lower concentrations.
As summarised in Figure 3, most of the phenolic compounds found in tamarillo might be
synthesised from the shikimic acid route. In general, phenolic compounds in plants are synthesised
from this route [26], where carbohydrate precursors are converted to phenylalanine and then to trans-
cinnamic acid, p-coumaric acid and dihydroflavonols. With the addition of hydroxyl groups and
sugars and methylation, these compounds are further derived to form various secondary phenolic
compounds, which were observed in tamarillo from the current study. For example, addition of sugar
molecules to the flavonol skeletons produce kaempferol-3-rutinoside, rutin and isorhamnetin-3-
rutinoside (Figure 3), and these were detected in this study (Table 1).
Chlorogenic acid, which is an ester formed between caffeic acid and quinic acid (Figure 3),
functions as an intermediate in lignin biosynthesis. In Solanaceous species which include eggplant,
potato and tomato, chlorogenic acid has been reported as the main soluble phenolic compound [27],
which was also observed as the dominating phenolic compound in the current study. The
concentrations of chlorogenic acid in New Zealand tamarillos was significantly higher than that in
tomato pulp, which ranged from 24.58 to 53.10 mg/100 g DW [28]. Variations in genetic traits and
physiological characteristics of plants (e.g., height of the tree and its respective absorption of sunlight)
may explain the differences in chlorogenic acid between these two species. According to Clé, Hill,
Niggeweg, Martin, Guisez, Prinsen and Jansen [29], significantly higher levels of soluble phenolics
are found in plants when grown under natural sunlight. With the strong UV light of New Zealand,
this may have helped in producing higher amounts of phenolic compounds to those sourced from
other countries. Ripeness of the fruit is also known to influence the production of chlorogenic acid
[30]. According to Onakpoya, Spencer, Thompson and Heneghan [31], intakes of foods containing
high levels of chlorogenic acid can result in significant reductions in systolic and diastolic blood
pressures. Health benefits of chlorogenic acid include anti-diabetic, anti-carcinogenic, anti-
inflammatory, anti-obesity and antioxidant properties [32], which would further increase the
bioactive potential for tamarillo as a functional ingredient.
Rutin, a natural flavone derivative, was the second-most abundant polyphenol identified in
New Zealand-grown tamarillos. Pure rutin carries yellow or yellow-green colour, which may explain
its higher presence in Amber cultivars than in two other varieties, regardless of the tissue type. Rutin
is a powerful antioxidant, known for its capacity to strengthen the blood cell walls [33], with
antitumor, antibacterial and antiviral [34], antioxidant and anticarcinogenic properties [35].
Moreover, rutin has been reported to help in collagen production and to enhance utilisation of
vitamin C [36], which is present in significant amounts in tamarillo (25 mg/100 g) [37]. Jang, Wang,
Antioxidants 2020, 9, 169 14 of 21

Lee, Lee and Lim [38] reported that kaempferol-3-rutinoside, known as a powerful α-glucosidase
inhibitor, owned anti-adipogenic potential, which may act as an anti-obesity agent. These authors
indicated that kaempferol-3-rutinoside showed inhibitory effects on adipogenesis to 48.2% without
cytotoxicity.
Caffeic and gallic acids possess biological functions associated with the modulation of
carcinogenesis. Gallic acid has been reported as a protector against oxidative damage and chemo-
preventive agents, resulting in the death of several tumour cell lines [5]. Foods derived from
flavonols—in this case, kaempferol—exhibit multiple health-promoting effects, such as inhibiting the
formation of cancer cells. Flavanols such as catechin and epicatechin, which were determined in
tamarillos for the first time, could contribute to increasing the overall antioxidant power of tamarillo.
Catechin is known to possess anticancer, anti-obesity, anti-inflammatory and neuroprotective
properties. With the presence of these phenolics, bioactive values of tamarillo are compatible to other
super fruits.
On top of health benefits, anthocyanins have raised a growing interest in improving postharvest
handling. With the presence of anthocyanins, lessening of overripening and longer shelf-life has been
observed [39], and this may explain the long season of tamarillo. Anthocyanins enhance the
antioxidant activity of fruits by suppressing reactive oxygen species (ROS), which will subsequently
slow down the overripening process [40]. By controlling the ROS burst, anthocyanins improve fruit
resistance to botrytis, minimising fungal growth [40]. Current findings support the previous
literature that delphinidin-based anthocyanins are the abundant structure of Solanaceous species,
including pepper, tomato, eggplant and potato [39]. Delphinidin-based anthocyanins have been
reported as inhibiting thrombosis and reducing vascular inflammation [41]. Additionally, by
preventing keratinocyte apoptosis, it is able to protect human skin against UV-B irradiance [39].
Cyanidin and its glycosides have been recognised as strong antioxidants. Their antioxidant
activity is stronger than that of vitamin E, vitamin C and resveratrol [25]. To date, bioavailability of
cyanidin-3-glucoside has been the most explored among these major anthocyanins [25]. Czank,
Cassidy, Zhang, Morrison, Preston, Kroon, Botting and Kay [42] have observed that within 48 h of
ingestion, the relative bioavailability of cyanidin-3-glucoside reached 12.38%, in which 5.37% was
excreted in urine and 6.91% was lost through breathing. This anthocyanin is easily absorbed into the
plasma [25]. Usually, cyanidin is concentrated in the skin of fruits [43], which was also observed in
the current study (Table 1). All of the peel samples showed higher amounts of cyanidin-3-rutinoside
than the pulps. Cyanidin-3-glucoside were only detected in Laird’s Large and Mulligan peels.
Therefore, anthocyanins extracted from the peels of these tamarillos could be used as food additives.
Similar applications have been made with grape skins to produce E163 [25]. The current findings also
raise an interest in using anthocyanin as a natural colorant to replace synthetic food dye.
In nature, cyanidin appears as a reddish-purple pigment, while delphinidin is seen as a purple
pigment. This can explain why the Amber cultivar had the least anthocyanin, compared to Laird’s
Large and Mulligan. Formation of flavylium cations are predominant in red pigments of
anthocyanins, and these are stable at acidic conditions [25]. The pH of golden-yellow and purple-red
tamarillo cultivars are 3.2–3.5 and 3.5–3.6, respectively [22], which are suitable for these anthocyanins
to be stable. The richer concentrations of anthocyanins in pulps than in peels might be due to the
presence of seeds, which have dark purple colours. Hurtado, Morales, González-Miret, Escudero-
Gilete and Heredia [44] reported that delphinidin-3-rutinoside owned the greatest ability in
capturing the 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), known as ABTS radical,
followed by cyanidin-3-rutinoside and then pelargonidin-3-rutinoside.
Light and temperature during the growth of tamarillos have been reported to influence the
anthocyanin metabolism. Anthocyanin biosynthesis is known to be stimulated by high light intensity
[45], and it is also affected by light quality in most plants. Guo, Han and Wang [46] reported that UV-
A radiation enhanced anthocyanin contents in tomatoes, compared to white light. Anthocyanin
accumulation in Solanaceae species is induced by low temperatures [47,48]. The strong UV light of
New Zealand and average temperatures of 10–18 °C from April to October, when tamarillo are being
Antioxidants 2020, 9, 169 15 of 21

grown, may explain the significantly higher amounts of anthocyanins in New Zealand tamarillos
compared to other sources.
Peels exhibited higher total phenolic contents, as determined by the Folin–Ciocalteu method,
and stronger antioxidant activity than the pulp of tamarillo. This seemed common in fruit, in general.
Polyphenols found in peels have been reported to possess antifungal and antibacterial properties
[12]. Extraction and purification of these compounds into a functional ingredient or additive may be
applicable for the food and pharmaceutical industries and to help with utilisation of by-product
(peels). High antioxidant activity of the peels is also related to higher colour stability [44], which is
helped by the presence of phenolic acids, organic acids, flavones, flavanones, flavonols and flavanols
[49,50].
The significant difference of antioxidant capacity observed across the cultivars and the tissues
could be related to their phenolic compositions. From the current study, Mulligan (purple) cultivars
showed the highest antioxidant capacity. Red cultivar showed higher antioxidant capacity than the
gold cultivar from New Zealand, which is in agreement with Lister et al. [1] who used the ABTS
assay. Greater antioxidant activity in purple-red tamarillos compared to the yellow species from New
Zealand were similar to Ecuadorian tree tomatoes, which were assessed by the ORAC assay [8] and
DPPH assay [24]. Many studies have demonstrated that tamarillo possesses higher antioxidant
capacity than commonly consumed fruits, such as apples, oranges, red grapes [8], kiwi fruit,
pineapple [1], tomatoes and cherry tomatoes [51], although the concentrations of phenolic
compounds in tamarillo are similar or lower than these fruits. This may be due to the presence of
certain phenolic compounds or other components which own stronger antioxidation properties; for
example, anthocyanins, carotenoids and phenolic acids. As discussed, the presence of phenolics,
anthocyanins, TPC and antioxidant activity of tamarillo suggest that this fruit, particularly the peels,
has a great potential to be utilised as a functional ingredient. Antioxidant activity of tamarillo has
also been evaluated using in vitro cancer cell lines. With 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium (MTT) assay, Ordóñez, Cardozo, Zampini and Isla [52] demonstrated that
tamarillo reduced oxidative stress in HepG2 liver cancer cells and provoked apoptosis in a dose-
dependent manner. Prevention of proliferation and viability of the HepG2 liver cancer cell line and
MDA-MB 231 breast cancer cell line have also been observed through MTT assay by Mutalib et al.
[11], where the most significant effect was seen with application of the crude ethanol extracts from
tamarillo containing 2.53 mg GAE/g DW of TPC. From our study, TPC in the aqueous ethanol extracts
of peels and pulps were much higher, as shown in Table 2 (6.79 to 22.3 mg GAE/g DW). Therefore,
we assume that similar cytotoxic effects may be expected if the tamarillo extract was to be applied to
HepG2 and MDA-MB 231 cell lines. Presence of higher TPC in peels compared to pulps may suggest
a new use of the peels, which are often discarded as by-products. Mutalib et al. [11] further reported
that the cytotoxic effects of tamarillo extracts against MDA-MB 231 and HepG2 cells were as good as
that of doxorubicin, which is a commercial anticancer drug. The strong correlation observed between
TPC and antioxidant activity (as identified by CUPRAC, FRAP and DPPH methods) indicated that
polyphenols were the major antioxidants in tamarillo regardless of the cultivars and tissues.
Chlorogenic acid, caffeic acid, catechin, ellagic acid, rutin and kaempferol and its glucosides
were among those with validated antioxidant activity demonstrated through in vitro and in vivo
studies. Treatments with chlorogenic acid of up to 40μM (14.2 μg/mL) have shown a protective ability
against t-BOOH-induced ROS generation in HepG2 cells (p < 0.05) [53] and H2O2-induced oxidative
stress in human HaCaT cells (p < 0.05) [54] and DNA damage in MCF-7 and MAD-MB-231-cultured
human breast cancer cells [55] and in human blood lymphocytes (p < 0.001) [56]. Through
supplementation of chlorogenic acid (5 mg/kg body weight) in diabetic rat models, reduction of lipid
hydroperoxide formation (p < 0.05) has also been reported [57]. From the current study, chlorogenic
acid found in aqueous ethanol extracts of peels and pulps from New Zealand tamarillos ranged from
113.2 to 133.6 and 26.4 to 36.3 μg/mL, respectively. Treatment effects were seen at lower chlorogenic
concentrations in the previous studies; hence, similar protective effects may be observed with these
tamarillos.
Antioxidants 2020, 9, 169 16 of 21

Extensive studies related to bioactive polyphenolic compounds and their related functions in
humans are found in the literature. These include inhibitory effects against oxidative stressors (t-
BHP, H2O2 and FeSO4) in PC12 cells by ellagic acid [58]; protective effects against DNA damage in
MCF-7 and MAD-MB-231-cultured human breast cancer cells by caffeic acid at 20 μM [55]; delay in
lipid oxidation and reduction in the formation of malondialdehyde activity (MDA) of platelets in
human plasma (p < 0.05) by catechin at 20 to 200 μg/mL [59]; reduction of ROS generation in H2O2-
treated APPswe cells (p < 0.05) by rutin at 50 to 100 nM [60]; prevention of concanavalin A (ConA)-
induced activation of T cell proliferation and nitric oxide and ROS formation in LPS-induced RAW
264.7 macrophage cells by kaempferol and its glycosides [61]. The data obtained from the current
study show that the tamarillos possessed higher amounts of those aforementioned compounds and
their effective concentrations. Therefore, similar effects may be observed if further in vitro and/or in
vivo studies were conducted with these tamarillos.
Similarly with the anthocyanins, high antioxidant capacity has also been demonstrated in
human colon cancer (Caco-2), human hepatocarcinoma (HepG2), human endothelial (EA.hy926) and
rat vascular smooth muscle (A7r5) cells with anthocyanins from bilberries and blueberries at low
concentrations (EC50 < 1 μg/L, nM range) [62]. An excellent antioxidant capacity has been observed in
the plasma of rats (p < 0.01) [63] and in the serum of humans (p < 0.05) [64] with increased intakes of
anthocyanins. Furthermore, treatment with anthocyanins extracted from purple-tomato peel at 52.5
μg/mL showed reduction in ovarian cancer cell lines (p < 0.05) [65]. Provided that the total
anthocyanins contents in the extracts of Laird’s Large peel and Mulligan peel were 87 and 144 μg/mL,
respectively (Table 1; unit converted to μg/mL), tamarillo peels are likely to show similar extents of
antioxidant activity. The respective ratio between these antioxidant compounds and anthocyanins
may further enhance the antioxidant capacity of the fruit, compared to the application of a single
compound to the cell lines. Further research of in vitro cell line-based assays and animal models
should be implemented for a deeper understanding of antioxidant activity in tamarillo.
To our best knowledge, this was the first study to measure the TPs and TPC of three tamarillo
cultivars from New Zealand separated into peel and pulp tissues. There was a slight difference
between TPC identified by the Folin-Ciocalteu method and TPs calculated from all phenolics
determined by LC-MS/MS. This discrepancy may be due to the Folin-Ciocalteu method measuring
the TPC-containing free phenolic groups only while the LC-MS/MS detects phenolics containing
glycosylated or ester-linked groups, such as rutin, kaempferol-3-rutinoside or isorhamnetin-3-
rutinoside. Additionally, the total concentrations of polyphenols calculated from the LC-MS/MS
results were only based on the identified compounds, while the TPCs were measured for the entire
extracts.
Antioxidants 2020, 9, 169 17 of 21

Phenylala

Ellagic

trans- Benzoic

Gallic
Ferulic

Caffeic
p-Coumaric

Chlorogenic
Shiki
mic
Kaempferol-3-
acid
p-Coumaroyl-

Chalc
Flavon

Isorhamnetin-3-

Ru
Dihydroflav
Catec
Epicate

Anthoc

Cyanidin-3-

Cyanidin-3-

Delphinidin-3- Pelargonidin -3-

Figure 3. Outline of phenolics and anthocyanins biosynthesis from phenylalanine in tamarillos.

5. Conclusions
Examination of antioxidant compositions and their capacities in three New Zealand tamarillo
cultivars showed that tamarillo peels possessed higher amounts of phenolic compounds, total
phenolic content and antioxidant activity than the pulps. Pulps had higher anthocyanins
Antioxidants 2020, 9, 169 18 of 21

concentration than the peels. For cultivars, Mulligan showed the highest antioxidant activity and the
highest amounts of phenolic and anthocyanin compounds, in general. Phenolic profile was
dominated by chlorogenic acid regardless of cultivars and tissues. The anthocyanin profiles of pulp
in all three cultivars were dominated by delphinidin-3-rutinoside. Antioxidant capacity of tamarillos
exhibited relatively high values and were strongly correlated with high total phenolic content.
Presence of these bioactive compounds highlight the potential of tamarillo for further utilisation in
food and pharmaceutical industries.
Further research in tamarillo to explore the interaction of these phenolics and anthocyanins with
other food components and how their antioxidant activity is influenced during food processing
would be advantageous. Tamarillo remains underutilised despite its bioactive components. It has a
relatively long fruit season, and they are relatively shelf-stable compared to other fruits (e.g.,
strawberries), which would be of great advantage.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1: Figure S1: Standard
curves of four anthocyanins detected in tamarillos using Agilent Chemstation Software (Agilent Technologies,
Australia) for LC-MS and Table S1: Method validation for quantification of polyphenols and anthocyanins
present in tamarillos, -

Author Contributions: Conceptualization, T.D. and M.Y.; methodology, C.P.; software, T.D. and C.P; validation,
T.D., C.P. and M.Y.; formal analysis, T.D.; investigation, T.D.; resources, T.D. and M.Y.; data curation, M.Y.;
writing—original draft preparation, T.D.; writing—review and editing, C.P and M.Y.; visualization, T.D. and
M.Y; supervision, M.Y. and project administration, M.Y. All authors have read and agreed to the published
version of the manuscript.

Funding: This research received no external funding

Acknowledgments: The authors would like to acknowledge the New Zealand Tamarillo Growers Association
for providing tamarillo fruits. The authors would like to thank the Riddet Institute for the doctoral scholarship
provided to the first author.

Conflicts of Interest: The authors declare no conflicts of interest.

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